Supporting Information Legends
Fig. S1. Reverse transcription PCR of Oma1-like genes encoded by the sugar beet
Rf1 locus. NK-198 (fertility restored line) and TK-81mm-O (maintainer line which is
devoid of any Rf but male fertile due to lack of S-orf from mitochondria) were used in
this experiment. Anthers, leaves, and roots of NK-198, and anthers of TK-81mm-O
were the tissues used in this analysis. Total cellular RNA was extracted with an
RNeasy Plant Mini Kit (Qiagen, Valencia, CA), and treated with RNase-free DNase I
(Takara Bio, Ohtsu, Japan). Complementary DNA was obtained using SuperScript III
First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Nucleotide sequences of
primers are 5'-TTTGGAAGGGATGAATTGGG-3' and 5'CATAACATCAGCTCGAGCTAA-3', which correspond to the first intron and the third
intron, respectively. A size marker is shown on the right (kbp). The target sequences
of these primers are conserved between NK-198 and TK-81mm-O copies. The PCR
protocol was 30 cycles of 94°C, 30 sec, 58°C, 30 sec, and 72°C, 2 min. PCR
products were electrophoresed in a 2% agarose gel. Integrity of the TK-81mm-O
amplicon was confirmed by nucleotide sequencing. No 0.6-kbp signal band was
seen when genomic DNA of TK-81mm-O was used as the template.
Fig. S2. Immunoblot analysis of sugar beet calli that were resistant to hygromycin.
Sugar beet suspension cells were infected with Agrobacterium transformed with the
bvORF20::flag transgene or the bvORF20L::flag transgene, then grown on a medium
containing hygromycin. Total cellular proteins from ten (lanes 1-10) and two (11-12)
calli, candidates having bvORF20::flag and bvORF20L::flag, respectively, were
electrophoresed in a 12% SDS-polyacrylamide gel. The blot was probed with antiFLAG antiserum (αFLAG). A size marker is shown on the left (kDa). B and N denote
blank and non-transgenic callus, respectively.
Fig. S3. Detection of a complex containing preSATP6. Size markers are shown to
the left of each image (kDa). A. Blue Native (BN) polyacrylamide gel electrophoresis
(PAGE) (3-12%) of mitochondrial proteins prepared from TK-81mm-O and TK81mm-CMS taproots. Proteins were prepared in the presence of digitonin (the
digitonin/protein ratio was 5.0 [w/w]). The gel was stained with Coomassie brilliant
blue (CBB). The obtained electrophoretic patterns were very similar between the two
sugar beet lines except for the 250 kDa signal band that was specific to TK-81mmCMS. A filled triangle indicates the 250 kDa signal band specific to TK-81mm-CMS.
B. Gel blot analysis of proteins separated in panel A. Anti-preSATP6 (αpreSATP6)
antiserum was used. A filled triangle indicates the 250 kDa signal band. C. Gel blot
analysis of proteins separated in panel A. Anti COXI antiserum (αCOXI) was used.
Open and shaded triangles indicate the 420 and 340 kDa signal bands (the two
super complexes containing mitochondrial Complex IV), respectively.
Fig. S4. Immunoblot analysis of immature anthers collected from TK-81mm-CMS.
Total cellular proteins were lysed in the presence of digitonin at a digitonin/protein
(w/w) ratio of 1.0, 2.5, 5.0, and 7.5. Gel blots were probed with αpreSATP6 or αCOXI.
Size markers are shown on the left (kDa). The 250 kDa complex detected with
apreSATP6 is stable in the digitonin/protein ratio range of 2.5 to 7.5. Note that the
digitonin/protein ratio employed for Figs. 2, 4, 6, 7, S3, S6 and S7 was 5.0.
Fig. S5. Light microscopic images of anther contents stained with Alexander's dye
(Alexander, 1969). Anthers were collected from NK-198. Collected anthers were
immediately squashed in a droplet of Alexander's dye to stain their contents. The
preparations were not incubated prior to observation. A, meiosis stage in which the
microspore mother cell undergoes meiosis (depicted as M in Fig. 5); B, early tetrad
stage in which meiosis is completed and four microspores firmly adhere to each
other (Ta); C, middle tetrad stage in which the four microspores begin to separate
(Tb); D, late tetrad stage in which the four microspores are clearly distinguished and
the callose wall becomes thinner (Tc); E, early microspore stage in which the callose
wall disappears, thereby releasing the microspores and a dense-stained globular
structure is apparent inside of the microspore (Sa); F, late microspore stage in which
the outer surface pattern becomes visible but the interior of the microspore is less
stained than at the mature stage (Sb); G, mature stage in which the pollen grain is
round, its surface exhibits a clear pattern, and interior of the pollen grain is deeply
stained (P).
Fig. S6. Immunoblot analysis of mitochondrial proteins from NK-219mm-CMS callus
expressing bvORF20::flag. Total mitochondrial proteins were lysed in the presence
of digitonin, and then subjected to 2D-PAGE consisting of BN-PAGE (3-12%) for the
first dimension (from left to right) and SDS-PAGE (4-12%) (from top to bottom). Gel
blots were probed with αpreSATP6 or αFLAG. Size markers are shown in the top
and left (kDa).
Fig. S7. Immunoblot analysis of mitochondrial proteins from NK-219mm-CMS callus
expressing bvORF20L::flag. Total mitochondrial proteins were lysed in the presence
of digitonin, and then subjected to 2DPAGE consisting of BN-PAGE (3-12%) for the
first dimension (from left to right) and SDS-PAGE (4-12%) (from top to bottom). Gel
blots were probed with αpreSATP6 or αFLAG. Size markers are shown in the top
and left (kDa).
Table S1. Nucleotide sequences of primers.